Single cell genome amplification and library building method and kit

Abstract

The invention provides a single-cell genome amplification library building method, which comprises the following steps of: splitting a single cell; dNA fragmentation: a cleavage product is subjected to DNA fragmentation with at least two restriction endonucleases, most recognition sites of the first restriction endonucleases are located in a high-CG region of the genome, and recognition sites of the second restriction endonucleases do not have CG; sequentially carrying out terminal repair A addition, linker addition and User enzyme treatment on the fragmented product; and library amplification: adding a sequence tag into the User enzyme treatment product, and carrying out DNA amplification, thereby completing single cell genome amplification and library construction in one step. The invention also provides a single cell genome amplification library building kit, which comprises a fragmentation enzyme combination, and the fragmentation enzyme combination comprises a second restriction enzyme without CG in first restriction enzyme recognition sites of which most recognition sites are located in a genome high CG region. According to the method and the kit, the sequencing success rate after library establishment can be greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a new single-cell genome sequencing method, kit and their applications. BACKGROUND

[0002] Genome sequencing technology has been widely used in basic research of life sciences and some corresponding transformational scientific application fields to obtain information of genome changes.

[0003] For multicellular organisms, during the process of cell division and differentiation, differences, i.e. heterogeneity of genetic information, will inevitably be gradually generated. The traditional research method is to complete detection on the basis of multicells, i.e. to process thousands of cells at a time, and the final signal value actually reflects the average level of genes in the group of cells, which will lead to the loss of expression heterogeneity information among different cells. If the single-cell sequencing method is used, the genetic information such as genome and transcriptome of different cells in the same cell group can be completely reflected.

[0004] Single cells need to be lysed and amplified first to obtain enough gene amount for library construction. The commonly used whole genome amplification methods at present include: multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). The advantage of MDA is that it has higher amplification efficiency, high fidelity and simpler experimental method. The characteristic of MALBAC method is that it has better amplification uniformity, but the amount of amplified DNA obtained is relatively small, and the amplification efficiency is relatively low. Studies have shown that MALBAC has advantages over MDA in single nucleotide site variation (SNV) and copy number variation (CNV) detection. Traditional high-throughput sequencing library construction mainly includes fragmenting the target genome by ultrasonic crushing or enzyme digestion after pre-amplification, and then a series of processes such as end repair, adapter addition, PCR amplification and magnetic bead purification. Overall, the single-cell amplification and library construction process is time-consuming and complicated to operate, and it is difficult to meet the timeliness requirements of some detections; or the quality of the constructed library is not high enough, and the subsequent sequencing success rate for CNV analysis is low. Therefore, a set of efficient and stable single-cell amplification and library construction method and kit has become an urgent need in molecular biology research. SUMMARY

[0005] In order to overcome the deficiencies of the prior art, the present application discloses a new single-cell sample genome amplification library construction method and kit, which can greatly improve the sequencing success rate.

[0006] The first aspect of the present application is a single-cell genomic amplification library construction method, comprising: single-cell lysis; DNA fragmentation; single-cell DNA fragmentation treatment of the lysis product with at least two restriction endonucleases, wherein the recognition site of the first restriction endonuclease is mostly located in the high-CG region of the genome, the recognition site of the second restriction endonuclease does not contain CG, and the combination of the first and second restriction endonucleases is used to obtain higher whole genome coverage; sequentially performing end repair plus A, adapter addition, and User enzyme treatment on the fragmentation product; and library amplification: adding a sequence tag to the User enzyme treatment product and performing DNA amplification, thereby completing the amplification and library construction of the single-cell genome in one step.

[0007] In the preferred method of the present application, the recognition site of the first restriction endonuclease is 5’…C ↓ CGG…3’, and the recognition site of the second restriction endonuclease is 5’…G ↓ CWGC…3’; specifically, wherein the first and second restriction endonucleases are MspI and ApekI, respectively.

[0008] In the preferred method of the present application, it further comprises performing copy number variation analysis on the constructed library.

[0009] The present application also provides a kit for directly constructing a single-cell genomic library with picogram-level DNA, comprising a fragmentation enzyme combination for fragmenting the DNA of a single cell, which combination comprises: a first restriction endonuclease, the recognition site of which is mostly located in the high-CG region of the genome, preferably the recognition site of which is 5’…C ↓ CGG…3’; and a second restriction endonuclease, the recognition site of which does not contain CG, preferably the recognition site of which is 5’…G ↓ CWGC…3’.

[0010] In the example of the kit, it further comprises: a single-cell lysis reagent; an end repair enzyme, a dVTP Mix, a T4 ligase, an adenine nucleotide triphosphate, a sequencing adapter, a User enzyme, an amplification reaction solution, a sequence tag, and a multifunctional buffer; and an instruction indicating that after single-cell lysis, DNA fragmentation is directly performed, and then the fragmentation product is sequentially subjected to end repair plus A, adapter addition, User enzyme treatment, and library amplification.

[0011] In the example of the kit, the amount of the reagents contained meets the following requirements: for the construction of a library of 80-120 single cells, MspI 120 μL and ApekI 120 μL are provided; an end repair enzyme 120 μL, a dVTP Mix 96 μL, a T4 ligase 60 μL, an adenine nucleotide triphosphate 30 μL, a sequencing adapter Adapter 120 μL, and a User enzyme 120 μL.

[0012] In another preferred embodiment of the kit, the single cell lysis reagent comprises a KOH component and a dithiothreitol component, both components are pre-formulated together as an aqueous solution or the two components aqueous solutions are mixed just before use, the molar ratio of the two components in the lysis solution is 1.5-3.0:1, preferably the molar ratio is 2.0-2.5:1.

[0013] Another aspect of the present application also relates to the use of the above-mentioned kit in performing the single cell genome amplification library construction method.

[0014] Another aspect of the present application relates to a single cell lysis solution, which comprises a KOH component and a dithiothreitol component, both components are pre-formulated together as an aqueous solution or the two components aqueous solutions are mixed just before use, the molar ratio of the two components in the lysis solution is 1.5-3.0:1, preferably the molar ratio is 2.0-2.5:1.

[0015] A preferred single cell lysis solution, wherein the concentration of the KOH component in the lysis solution is 150-250 mM, preferably 200 mM.

[0016] Another aspect of the present application relates to the use of the above-mentioned single cell lysis solution in single cell lysis.

[0017] The use of the single cell lysis solution of the present application also includes: the DNA released after single cell lysis is subjected to single cell genome amplification by MALBAC method; and, optionally, single cell genome library construction and copy number variation analysis.

[0018] In a preferred embodiment of the use of the single cell lysis solution of the present application, the single cell is fixed by 3,3'-dithiodipropionic acid bis(N-hydroxysuccinimidyl ester) and / or 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimidyl ester) as a cell fixation agent.

[0019] In a preferred embodiment of the use of the single cell lysis solution of the present application, the termination solution for single cell lysis is tris(hydroxymethyl)methylamine solution.

[0020] The present application also relates to the use of the combination of restriction enzymes MspI and ApekI in performing the single cell genome amplification library construction method of the present application or in preparing the kit of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Single enzyme digestion and double enzyme digestion results of single cell genomes are compared.

[0022] Figure 2 A urine sample cell fluorescence staining chart.

[0023] Figure 3 A sampling quality control chart of multiple single cell genome amplification products.

[0024] Figure 4 and Figure 5 The DNA release effects of the lysis method of this invention combined with different cell fixation methods were compared.

[0025] Figure 6 This is a diagram showing the results of single-cell CNV detection in human embryos using the one-step method of this invention.

[0026] Figure 7 This image shows the results of single-cell CNV isolated from human ascites fluid detected by the two-step method of this invention.

[0027] Figure 8 This image shows the results of single-cell CNV detection of CTCs in human blood using the two-step method of this invention. Detailed Implementation

[0028] This invention provides an integrated method and kit for single-cell amplification and library construction (referred to as the one-step method or one-step kit), which realizes the amplification and library construction of a single-cell genome in one step, meaning that the single-cell amplification product is the sequencing library. A single cell contains very little DNA, only about 5-10 pg, while conventional library construction requires starting DNA at the nanogram (ng) level. This kit, however, can use single-cell DNA, i.e., picogram (pg) level starting DNA, as a template to achieve one-step amplification and library construction, and the sequencing success rate of the constructed library is >90%.

[0029] In another aspect, this invention provides a single-cell lysis buffer and lysis method, which, combined with existing single-cell genome amplification and library preparation techniques, can significantly improve library preparation quality. For single-cell sequencing of clinical tumor samples, it can increase sequencing success rate to >90%.

[0030] Successful sequencing is defined as sequencing data quality meeting the requirements for copy number variation analysis, i.e., the MAPD value (a standard for evaluating data quality) is below 0.45.

[0031] Besides tumor cells, both aspects of this invention are also applicable to library construction and subsequent gene sequencing analysis of various types of isolated single cells. For example, the one-step kit of this invention is also suitable for single-cell genomic library construction and subsequent sequencing of embryonic samples. The one-step kit is simpler to operate, less time-consuming, and offers high sequencing uniformity and coverage.

[0032] The single cell used in the present application is a cell suspension formed after processing tissue cells or a sample containing target trace cells obtained by cell separation. The sample can be derived from a human, and the target cell is usually a cell with a rare content in the human body but with important research value, such as a peripheral blood circulating tumor cell, a tumor cell exfoliated in a body fluid (including but not limited to urine, serous cavity effusion, various lavage fluids, various puncture fluids, etc.), and a tumor tissue cell. The cell separation is a conventional single cell separation technology, such as a capillary separation technology, a microdissection technology, and flow cytometry. However, the single cell can also be a single cell of an embryo sample or other single cells. The cell separation is a conventional single cell separation technology, such as a micromanipulation technology, a microdissection technology, and flow cytometry. However, the single cell can also be a single cell of an embryo sample or other single cells. For a tumor single cell, especially a rare tumor cell from a body fluid, a cell fixation step is usually required in the acquisition process to maintain the stability of antigens and the integrity of nucleic acid substances in the process of cell labeling, screening, and separation. When the single cell genome amplification and / or library construction method of the present application is implemented, for cells that need to be fixed, DSP (3,3'-dithiodipropionic acid di(N-hydroxy succinimide ester)) and / or SPDP (3-(2-pyridyl disulfide) propionic acid N-hydroxy succinimide ester)) are preferably used as cell fixatives for fixation.

[0033] The main difference between the one-step method for single cell genome amplification and library construction of the present application and the traditional two-step method (specifically, the use of MALBAC genome amplification for library construction) is that the genome pre-amplification and genome amplification steps are omitted, and the single cell lysis product is directly subjected to DNA fragmentation with a fragmentation enzyme.

[0034] The steps of the single cell amplification and library construction integrated method of the present application include, in sequence, cell lysis, DNA fragmentation plus A, end repair, adapter addition, User enzyme treatment, and library amplification. The DNA fragmentation of the single cell is achieved using a combination of two restriction enzymes (for example, MspI+ApekI double digestion).

[0035] In one embodiment of the present application, the kit for single cell genome integration amplification and library construction includes a lysis buffer, a lysis enzyme, fragmentation enzymes MspI+ApekI, an end repair enzyme, dVTP Mix, a T4 ligase, adenosine triphosphate (ATP), a sequencing adapter, a User enzyme, an amplification reaction solution, a sequence tag (Barcode), and a multifunctional buffer (commonly used in the steps of fragmentation, end repair, and adapter addition).

[0036] The recognition site of MspI is 5'…C ↓ CGG…3', and the recognition site of ApekI is 5'…G ↓CWGC…3’.

[0037] The present application performs electronic enzyme cutting on single-cell genomes from rare tumor cells in body fluids under the same conditions, and compares the proportion of 0-300 bp fragments. It is found that the proportion is 52.4% or 52.05% when MspI or ApekI single enzyme cutting is performed, and the proportion is 63.75% when double enzyme cutting is performed.

[0038] The present application further uses agarose gel electrophoresis to detect the integrity of DNA after enzyme cutting, and compares the enzyme cutting results of single-cell genomes from rare tumor cells in body fluids by MspI single enzyme cutting and MspI+ApekI double enzyme cutting (see Figure 1 ). It is found by comparison that the main band of genomic DNA after single enzyme cutting does not change significantly, only a small part of DNA is cut off, and most of the DNA is still complete. Under the same conditions, after double enzyme cutting, the DNA distribution is dispersed, and there is no main band, which shows that the fragmentation effect of double enzyme cutting on genomic DNA is greatly improved, and the broken DNA has good uniformity.

[0039] Therefore, in order to perform the one-step single-cell genome amplification library of the present application, a DNA fragmentation enzyme with recognition sites concentrated in the high CG region of the genome and a DNA fragmentation enzyme without CG in the recognition site can be combined to obtain higher whole genome coverage.

[0040] The cell lysis reagent of another aspect of the present application includes a lysis solution and a lysis termination solution. The lysis solution contains an aqueous solution of KOH and dithiothreitol (DTT). The termination solution is tris(hydroxymethyl)methylamine. In a preferred embodiment of the present application, such as Example 2, the lysis solution is prepared and used as follows:

[0041] ① Preparation of single-cell lysis solution

[0042] KOH, 200 mM 11 μL

[0043] DTT, 1 M 1 μL

[0044] Total, 12 μL

[0045] Note: The prepared lysis solution should not be stored for more than three months;

[0046] ② Add 1.5 μL of single-cell lysis solution to the PCR tube containing single cells;

[0047] ③ Place the sample PCR tube in a PCR instrument at 65°C for 10 min; set the PCR instrument lip to 90°C;

[0048] ④ Add 1.5 μL of termination solution (200 mM tris(hydroxymethyl)methylamine) per tube, mix gently, and centrifuge. Continue to place on ice.

[0049] The lysis solution of the present application can more effectively lyse cells and fully release DNA therein, thereby significantly improving the quality of single-cell whole genome amplification.

[0050] The lysis solution and lysis method of the present application can be used in existing single-cell genome amplification technology, for example, to replace the enzyme lysis solution of existing single-cell genome amplification and library construction kits on the market, to improve the quality of subsequent pre-amplification and amplification, thereby improving the quality of subsequent library and sequencing success rate. Of course, those skilled in the art can easily understand that the lysis solution of the present application can also be used for the cell lysis step of the one-step method single-cell genome amplification library construction process of the present application if necessary.

[0051] The lysis solution of the present application can be used in existing two-step method single-cell genome amplification and library construction, for example, can include:

[0052] The lysis solution of the present application is added to the isolated single cell, and the single cell is lysed in a preheated PCR instrument according to the lysis method of the present application;

[0053] According to MALBAC, the genome is pre-amplified and genome amplified, the amplification product is purified by magnetic beads (such as AMPure XP magnetic beads), and the purified product is detected by Qubit for concentration, and the concentration is required to be greater than 10 ng / μL;

[0054] Optionally, as needed, single-cell genome amplification DNA quality control primers can be used for PCR testing, and bands between 100-500 bp are detected by agarose gel electrophoresis, and if there are 2-3 bands, it is a qualified amplification product, so as to quickly screen out sample single-cell whole genome amplification products meeting the library construction requirements;

[0055] DNA fragmentation is performed by enzyme breaking method;

[0056] The fragmented product is end-repaired and A-tailed;

[0057] The adapter is ligated, and the product is purified; and

[0058] Library amplification.

[0059] Examples

[0060] The single cells obtained from the urine sample were subjected to single-cell amplification and library construction by traditional two-step method (MALBAC amplification + library construction), two-step method using the cell lysis solution of the present application, and one-step method of the present application, and then subjected to sequencing by MGISEQ2000, as follows.

[0061] Obtaining single cells from urine:

[0062] 1) After the urine sample 50 mL was inverted and mixed uniformly, it was poured into a 50 mL centrifuge tube, centrifuged at 600 g for 10 min.

[0063] 2) Pour off the supernatant at a constant speed, add 1 mL of PBS for resuspension, and transfer to a 1.5 mL centrifuge tube; centrifuge,

[0064] 3) Centrifuge at 400g for 5 min, and remove the supernatant with a pipette, and add 100 μL of PBS for resuspension of the cells;

[0065] 4) Liquid-based preparation of a slice: load the cell suspension onto a slice, and let it settle for 10 min, and put it into a slice shaker for 5 min;

[0066] 5) Fix with cell fixative DSP (1-2 mM) and SPDP (1-2 mM) in a PBS buffer system for 35 min;

[0067] 6) Remove the fixative, and add 100 μL of a termination solution, and terminate the cross-linking at room temperature;

[0068] 7) After washing, block with a 2% fetal bovine serum-bovine serum albumin (2% FBS-BSA) solution at room temperature for 1 h.

[0069] 8) Add 100 μL of a membrane breaker, and incubate at room temperature for 15 min;

[0070] 9) After washing with PBS, block with a 2% fetal bovine serum-bovine serum albumin (2% FBS-BSA) solution at room temperature for 1 h.

[0071] 1 h;

[0072] 10) HK2, panCK-PE, and CD45-APC monoclonal antibodies are diluted to a use concentration using a PBS buffer at a dilution ratio of 1:100, the blocking solution is discarded, and a mixed solution of the antibodies is added, and incubated overnight at 4°C;

[0073] 11) Discard the antibody solution, and wash three times with a PBS buffer;

[0074] 12) Add 100 μL of a blocking solution, and block for 1 h at room temperature for the second time;

[0075] 13) Fluorescently labeled secondary antibodies are diluted to a use concentration using a PBS buffer at a dilution ratio of 1:400, the blocking solution is discarded, and a diluted secondary antibody solution is added, and incubated for 1 h at room temperature;

[0076] 14) Discard the secondary antibody solution, and wash three times with a PBS buffer, add a DAPI cell nucleus dye at a concentration of 1x, and incubate for 10 min at room temperature;

[0077] 15) Discard the secondary antibody solution, and wash three times with a PBS buffer, add a DAPI cell nucleus dye at a concentration of 1x, and incubate for 10 min at room temperature;

[0078] 14) Discard the secondary antibody solution, and wash three times with a PBS buffer, add a DAPI cell nucleus dye at a concentration of 1x, and incubate for 10 min at room temperature;

[0079] 15) Wash with PBS buffer for three times, and use single cell image analysis automatic separator to collect fluorescence image and

[0080] analysis, Figure 2 for one of the partial images;

[0081] 16) Recycle 150 HK2+ / panCK+ / CD45- / DAPI+or

[0082] HK2- / panCK- / CD45+ / DAPI+single cells by means of micro-operation platform.

[0083] The above obtained single cells are respectively taken 50 single cells to carry out single cell amplification and library construction by means of one-step method (Example 1) of the present application, two-step method (Example 2) combined with lysing solution of the present application and traditional two-step method (Comparative Example 1).

[0084] Example 1: One-step method for amplification and library construction

[0085] 1. Add 0.4 μL protease (NEB), 0.6 μL lysis buffer (Triton-X 100, sigma, Tris-EDTA, sigma, KCl, sigma) in single cell sample (4-5 μL), 55℃, 10 min; 80℃, 10 min, lyse single cell to release genomic DNA;

[0086] 2. DNA fragmentation: directly add 1 μL fragmentation enzyme A (MspI, Thermo), 1 μL fragmentation enzyme B (ApekI, NEB), 1.8 μL multifunctional buffer (Tango buffer (10X), Thermo), 9.2 μL H2O in lysis product, 37℃, 15 min, 75℃, 15 min, 80℃, 20 min;

[0087] 3. End repair and A addition: add 1 μL end repair enzyme (Klenow Fragment 3'→5'exo-, Abclonal), 0.2 μL multifunctional buffer (Tango buffer (10X), Thermo), 0.8 μL dVTP Mix (dATP, dGTP, dCTP, NEB) in fragmentation product, 37℃, 40 min, 75℃, 15 min;

[0088] 4. Ligation: Add 0.5 μL T4 Ligase (T4 DNA Ligase (High Conc.), Thermo), 0.5 μL Multi-purpose Buffer (Tango Buffer (10X), Thermo), 0.25 μL ATP (ATP, Thermo), 1 μL sequencing adapter, 3 μL H2O to the product of step 3, 22°C, 20 min, 65°C, 10 min;

[0089] 5. User enzyme treatment: Add 1 μL User enzyme ( USER Enzyme, NEB) to the product of step 4, 37°C, 30 min;

[0090] 6. Library amplification: Add 30 μL amplification reaction solution (2X kapa hot start Mix, KAPA), 4 μL sequence tag Barcode to the product of step 5, the reaction condition is 98°C, 2 min; 22 cycles (98°C, 20 s; 60°C, 30 s; 72°C, 60 s), 72°C, 5 min;

[0091] 7. Purify the library amplification product with AMpure XP magnetic beads;

[0092] 8. Quantify the PCR product with dsDNA HS Assay Kit.

[0093] Example 2: Two-step method for amplification and library construction using the lysis solution of the application

[0094] A. Release genomic DNA by lysing single cells: Add 1.5 μL single cell lysis solution (see preparation of single cell lysis solution and related conditions above) to each single cell sample, 65°C, 10 min; add 1.5 μL stop solution.

[0095] After the lysis step, use the single cell genomic amplification kit (Single Cell Whole Genome Amplification Kit, Yikang Gene, KT110700150) for genomic amplification (steps B-C), then use the single cell genomic library construction kit (MGICare Chromosome Copy Number Variation Detection Kit, Huada Gene, 1000012919 (96RXN)) for single cell whole genome library construction (steps G-K), the specific steps are as follows:

[0096] ​​​B. Single-cell genomic DNA pre-amplification: 30 μL pre-amplification buffer and 1 μL pre-amplification enzyme were mixed, and 30 μL reaction solution was added to the single-cell sample; the reaction condition was 94℃, 3 min; 8 cycles (20℃, 40 s; 30℃, 40 s; 40℃, 30 s; 50℃, 30 s; 60℃, 30 s; 70℃, 4 min; 95℃, 20 s; 58℃, 10 s);

[0097] C. Single-cell genomic DNA amplification: 30 μL amplification buffer and 1 μL amplification enzyme were mixed, and 30 μL reaction solution was added to the single-cell sample; the reaction condition was 94℃, 30 s; 17 cycles (94℃, 20 s; 58℃, 30 s; 72℃, 3 min);

[0098] D. PCR test using single-cell genomic amplification DNA quality control primer: three pairs of quality control primers were used in a PCR system 95℃, 3 min, 30 cycles (95℃, 30 s; 60℃, 30 s; 72℃, 30 s); 72℃, 5 min;

[0099] E. The PCR result was detected by gel electrophoresis, and the sampling result was shown in the electrophoretogram of the first 8 samples in Figure 3 , and 2 or more bands between 100-500 bp were determined as high-quality amplification products.

[0100] F. Amplification product purification: the amplification product was purified using AMPure XP beads;

[0101] G. Single-cell whole-genome amplification product fragmentation: 50 ng of purified DNA sample was reacted in 10 μL fragmentation and repair mixture, 37℃, 15 min; 65℃, 15 min;

[0102] H. Linker ligation: 33 μL ligation buffer and 2 μL ligase were mixed and added to the sample with the corresponding labeled linker, and reacted at 23℃ for 20 min;

[0103] I. Magnetic bead purification of ligation product;

[0104] J. Library amplification: 2 μL PCR primer mixture was added to 25 μL PCR reaction solution, and then added to the purified ligation product, and the reaction condition was 95℃, 3 min; 7 cycles (98℃, 15 s; 56℃, 15 s; 72℃, 30 s), 72℃, 5 min;

[0105] K. Magnetic bead purification of library amplification product;

[0106] L. PCR product quality control: using The dsDNA HS Assay Kit was used to quantify the PCR purified products, and the Agilent 2100 Bioanalyzer was used to detect the length distribution range of the PCR products.

[0107] Comparative Example 1: MALBAC + single cell library construction method

[0108] The same two kits as in Example 2 were used for single cell lysis and whole genome amplification (steps A-C) and library construction (steps E-I), and the specific steps are as follows:

[0109] A. Lysis of single cells to release genomic DNA: lysis enzyme, 55°C, 3 hours; 85°C, 15 min;

[0110] B. Single cell genomic DNA pre-amplification: mix 30 μL of pre-amplification buffer and 1 μL of pre-amplification enzyme, and add 30 μL of the reaction solution to the single cell sample; the reaction conditions are 94°C, 3 min; 8 cycles (20°C, 40s; 30°C, 40s; 40°C, 30s; 50°C, 30s; 60°C, 30s; 70°C, 4 min; 95°C, 20s; 58°C, 10s);

[0111] C. Single cell genomic DNA amplification: mix 30 μL of amplification buffer and 1 μL of amplification enzyme, and add 30 μL of the reaction solution to the single cell sample; the reaction conditions are 94°C, 30s; 17 cycles (94°C, 20s; 58°C, 30s; 72°C, 3 min);

[0112] D. Purification of amplification products: purification was performed using AMPure XP beads;

[0113] E. Fragmentation of single cell whole genome amplification products: take 50 ng of purified DNA sample and react in 10 μL of fragmentation and repair mixture, 37°C, 15 min; 65°C, 15 min;

[0114] F. Linker ligation: mix 33 μL of ligation buffer and 2 μL of ligation enzyme, and add to the sample with the corresponding labeled linker, 23°C, 20 min;

[0115] G. Magnetic bead purification of ligation products;

[0116] H. Library amplification: mix 2 μL of PCR primer solution in 25 μL of PCR reaction solution, and add to the purified ligation products, the reaction conditions are 95°C, 3 min; 7 cycles (98°C, 15s; 56°C, 15s; 72°C, 30s), 72°C, 5 min;

[0117] I. The library amplification products were purified using magnetic beads;

[0118] J. PCR product quality control: using The dsDNA HS Assay Kit was used to quantify the purified PCR products, and the length distribution range of the PCR products was detected using an Agilent 2100 Bioanalyzer.

[0119] The amplification product of step C in Comparative Example 1 was also subjected to quality control using the same steps as D and E in Example 2. The sampling and testing results are shown in [Figure 1]. Figure 3 The electrophoresis images of the last 15 samples in this study show that, compared to Example 2, a significantly smaller proportion of the samples are high-quality amplification products.

[0120] Compared to other lysis buffers, the lysis method used in Example 2 can more effectively lyse cells and fully release the DNA, which helps to significantly improve the quality of single-cell whole genome amplification products, thereby improving the quality of the subsequently constructed library and thus increasing the sequencing success rate of single-cell copy number variation analysis.

[0121] The single-cell gene libraries obtained in Examples 1, 2, and Comparative Example 1 were all sequenced using the MGICare sequencing reaction kit and the MGISEQ 2000 sequencer. The criterion for successful sequencing was that the sequencing data quality met the requirements for copy number variation analysis, i.e., the MAPD value (a standard for evaluating data quality) was below 0.45.

[0122] The table below compares the amplification and library preparation time and sequencing success rate of each method. Sequencing success rate is related to the quality of the preceding amplification; this success rate result is consistent with... Figure 3 The results of the sampling inspection of the amplified products shown are largely consistent.

[0123]

[0124] This application further explores the effects of the single-cell lysis buffer and lysis method of the present invention on samples with different cell fixation methods, mainly reflected by the number of quality control bands of the single-cell DNA amplification products after lysis.

[0125] Fresh urine samples were collected from healthy volunteers.

[0126] ① Divide urine cells into two groups: group A is fixed with PFA (paraformaldehyde, a commonly used fixative), and group B is fixed with DSP+SPDP. ② Obtain single cells, lyse the cells using the lysis method of this invention, and then perform single-cell genome amplification using the MALBAC method. ③ Use 22 pairs of quality control primers to perform PCR quality control on the amplification products of groups A and B, respectively.

[0127] The quality control results of DSP+SPDP fixation + the pyrolysis method of this invention are shown in the figure. Figure 4; PFA fixation + the control results of the splitting method of the application are shown in Figure 5 Here, 22 pairs of primers are used to "sample check" 22 chromosomes respectively, and the more the number of bands, the better the amplification quality. From the results of the two groups, it can be seen that the number of bands of the DSP + SPDP group is 20, and the number of bands of the PFA group is 12. It shows that the splitting method of the application combined with the DSP + SPDP fixing group is unexpectedly better. Further experiments show that the effect of DSP or SPSP alone as a fixing agent is similar. Figure 4 Figure 5 It can be seen that the DSP + SPDP group has 20 obvious bands, while the PFA group has only 12 bands. It shows that the splitting method of the application combined with the DSP + SPDP fixing group is unexpectedly better. Further experiments show that the effect of DSP or SPSP alone as a fixing agent is similar.

[0128] Example 3: One-step kit for human embryo single cell genomic library construction

[0129] 5 human embryo single cells were subjected to single cell lysis, library construction according to the operation steps of the one-step method of the application described in Example 2; PCR product quality detection: using dsDNA HS Assay Kit to quantify the product after PCR purification, the concentrations were 13.9 ng / μL, 13.5 ng / μL, 27.2 ng / μL, 31.6 ng / μL, 5.01 ng / μL, respectively.

[0130] Each constructed library was sequenced using the Illumina sequencing platform for single cell CNV analysis. The results showed that all 5 single cells were successfully sequenced, with a success rate of 100%, and the CNV results of one of them are shown in Figure 6 .

[0131] Example 4: The splitting solution of the application is used for amplification and library construction of single cells from pleural fluid, ascites and cerebrospinal fluid.

[0132] Collect pleural fluid, ascites and cerebrospinal fluid samples, and separate panCK+ / HK2+ / CD45- / DAPI+ single cells according to the steps of the foregoing "Method for constructing single cell genomic library" Obtaining single cells from urine " and according to the operation steps of the two-step method of the application described in Example 2, the cells were lysed, the single cells were amplified, and the library was constructed.

[0133] Library sequencing was performed using Huada MGISEQ2000, and the sequencing success rates of single cell libraries from various sources were as follows: pleural fluid 93.0% (199 / 214), ascites 93.5% (330 / 353), and cerebrospinal fluid 92.0% (23 / 25). Single cell CNV analysis was performed, and the analysis results of the ascites sample are shown in Figure 7 .

[0134] Example 5: The splitting solution of the application is used for single cell library construction of circulating tumor cells (CTC).

[0135] a) Collect 5 mL of peripheral blood from a patient with metastatic lung cancer in an EDTA anticoagulant tube; ​​

[0136] b) Add 20% CD36 cocktail (10 μL / mL whole blood) to whole blood, mix well, and incubate at room temperature for 10 min.

[0137] c) Add 3 times the volume of 2% FBS-HBSS and mix well;

[0138] d) Add density gradient centrifuge solution through the hole in the middle of the Sepmate tube septum, trying to avoid air bubbles. The centrifuge solution needs to be brought to room temperature.

[0139] e) Carefully add the blood and 2% FBS-HBSS mixture along the wall of the Sepmate tube to prevent mixing between layers;

[0140] f) Centrifuge at room temperature, 1200g, for 10 minutes. Centrifuge brake: acceleration 9, deceleration 6;

[0141] g) Pour out the supernatant;

[0142] h) Centrifuge again, 600g, for 8 minutes. Centrifuge brakes: acceleration 6, deceleration 6;

[0143] i) Discard the supernatant, add 1 mL of erythrocyte lysis buffer, and lyse the erythrocytes for 10–15 min.

[0144] j) Centrifugation: 300g, 5min;

[0145] k) Discard the supernatant, add 1 mL of HBSS to resuspend, and centrifuge at 300 g for 5 min;

[0146] l) Remove the supernatant, add 600 μL of PBS, and count;

[0147] m) Add 15 μL of cell fixative DSP / SPDP, mix quickly, add to the slide preparation chamber and let stand for 30 min (less than 500,000 cells per slide), centrifuge at 900 rpm for 5 min;

[0148] n) Add 100 μL of stop solution and incubate for 10 min;

[0149] o)“ Obtaining single cells from urine Immunofluorescence staining and image acquisition were performed in the relevant steps.

[0150] p) Single cells of HK2+ / panCK+ / CD45- / DAPI+ were recovered using a micromanipulation platform and transferred to 5 μL of cell lysis buffer;

[0151] q) Perform cell lysis, single-cell amplification, and library construction according to the two-step operation steps of the present invention as described in Example 2.

[0152] The library was sequenced by Huada MGISEQ2000, and the success rate of single cell sequencing was 90.8% (138 / 152). Part of the single cell CNV analysis results are shown in Figure 8 .

Claims

1. A method for single cell genomic amplification library construction, comprising: single cell lysis; DNA fragmentation: the lysis product is treated with at least two restriction enzymes for single cell DNA fragmentation, wherein the recognition sites of the first restriction enzyme are mostly located in the high CG region of the genome, and the recognition sites of the second restriction enzyme do not contain CG, and the combination of the first and second restriction enzymes is used to obtain higher whole genome coverage; end repair plus A, adapter ligation, and User enzyme treatment are sequentially performed on the fragmented product; and library amplification: sequence tags are added to the User enzyme treatment product and DNA amplification is performed, thereby completing the amplification and library construction of the single cell genome in one step.

2. The method of claim 1, wherein the recognition site of the first restriction enzyme is 5'...C ↓ CGG... 3' and the recognition site of the second restriction enzyme is 5'...G ↓ CWGC... 3'; in particular, wherein the first and second restriction enzymes are Mspl and ApekI, respectively.​​ 3. The method of claim 2, wherein the DNA fragmenting step comprises: 0.5-1.5 μL MspI, 0.5-1.5 μL ApekI, and enzyme digestion buffer are added to the lysis product of the single cell for enzyme digestion; and / or the enzyme digestion is maintained at 37°C for 15 minutes, 75°C for 15 minutes, and 80°C for 20 minutes.

4. The method of any one of claims 1-3, further comprising performing copy number variation analysis on the constructed library.

5. A kit for directly constructing a single cell genomic library with picogram level DNA, comprising a fragmentation enzyme combination for fragmenting the DNA of a single cell, which combination comprises: a first restriction enzyme, whose recognition site is located mostly in the high CG regions of the genome, preferably whose recognition site is 5'...C ↓ CGG... 3'; and a second restriction enzyme which recognizes a site which does not have a CG, preferably which recognizes a site of 5'...G ↓ CWGC... 3'.

6. The kit of claim 5, further comprising: a single cell lysis reagent; an end repair enzyme, a dVTP Mix, a T4 ligase, an adenine nucleotide triphosphate, a sequencing adapter, a User enzyme, an amplification reaction solution, a sequence tag, and a multifunctional buffer; and an instruction indicating that after lysis of the single cell, DNA fragmentation is directly performed, followed by end repair plus A, adapter ligation, User enzyme treatment, and library amplification of the fragmented product.

7. The kit of claim 6, wherein the amount of reagents contained is consistent with: for the construction of a library of 80-120 single cells, 120 μL of MspI and 120 μL of ApekI are provided; 120 μL of end repair enzyme, 96 μL of dVTP Mix, 60 μL of T4 ligase, 30 μL of adenine nucleotide triphosphate, 120 μL of sequencing adapter, and 120 μL of User enzyme.

8. The kit of claim 6, wherein the amount of reagents contained is consistent with: for the construction of a library of 80-120 single cells, 48 μL of single cell lysis enzyme and 72 μL of lysis buffer are provided; 120 μL of MspI and 120 μL of ApekI; 120 μL of end repair enzyme, 96 μL of dVTP Mix, 60 μL of T4 ligase, 30 μL of adenine nucleotide triphosphate, 120 μL of sequencing adapter, 120 μL of User enzyme, 1200 μL of amplification reaction solution, 240 μL of sequence tag, and 300 μl of multifunctional buffer. ​ ​ 9. The kit of claim 6 or 7, wherein the single cell lysis reagent comprises a KOH component and a dithiothreitol component, either pre-formulated together as an aqueous solution or two aqueous solutions of the components are mixed just before use, the molar ratio of the two components in the lysis solution being 1.5-3.0:1, preferably 2.0-2.5:

1.

10. Use of the kit of any one of claims 6-9 for performing the method of single cell genome amplification library building of any one of claims 1-5.

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